The present invention relates generally to nanometer-scale apertures and new techniques for fabrication thereof.
Nanometer-scale pores, channels, and slits (collectively called nanoapertures herein) have a variety of physical and chemical properties which can make them useful in a variety of applications. Examples include separation of chemical, isomeric, or isotopic species, control of surface diffusion, electrokinetic fluidic devices, and DNA sequencing. Suitable nanopores and nanochannels must have shapes which are defined and fabricated with dimensions typically of a few nanometers, with tolerances of a nanometer or less, and chemical specificity at the atomic level. This is necessary so that the proper types of interaction take place between the structure of the nanopore or nanochannel and the atomic or molecular entities involved. Such interactions can take the form of xe2x80x9cwettingxe2x80x9d or xe2x80x9cnon-wettingxe2x80x9d down to the level of sub-molecular moieties. Thus, nanopores and nanochannels can be made sensitive to not only the size, but also the shape and chemical structure of molecules. DNA sequencing will be used as a recent illustration of the utility of nanopores. Sequencing of the DNA molecule offers tremendous promise for medical and biotechnological applications of the future. To allow routine diagnostic use of this technique, however, it will be necessary to greatly decrease the time and expense required to decode a particular strand of DNA.
A new approach to DNA sequencing is being developed. The principle is to pull a single strand of double-stranded DNA through a miniscule aperture. An ionic current is used to pull the DNA strand. The DNA unzips one base pair at a time as the single strand threads through the aperture. As a result, when they pass through the aperture, they block the aperture in a manner characteristic of their molecular shape and local charge densities. A reduction thus appears in the ionic current which is characteristic of the molecular species passing through the aperture.
DNA has four base pairs, the amino acids adenine, thymine, guanine, and cytosine. Because these base pairs have different sizes and shapes, the amount of blockage observed is different for each species of base pair. By monitoring the current as the DNA strand is pulled through the aperture, direct sensing of the composition and ordering of the DNA strand can be accomplished.
The sequencing technique described above is still in the earliest stages of development, but offers promise for sequencing base pairs at a rate perhaps as high as 1000 base pairs per second. A practical apparatus with a reasonable amount of parallelism could then read an entire human genome in less than a day. This data could then be searched and decoded to reveal a wide range of genetic traits, including hereditary diseases, genetic tendencies toward disease and genetically-determined metabolic factors relative to pharmacological therapeutical choices.
Initial studies were based on the use of organic ion channels as the sequencing aperture. Ion channels are the xe2x80x9cporesxe2x80x9d in cellular membranes which regulate the flow of chemicals in and out of the cell. In particular, a channel called alpha haemolysin was used. The effective diameter of such ion channels is about 1.5 nm.
A cell membrane comprising such a channel was used to separate two compartments containing a potassium ion solution. When DNA strands were introduced into one of the compartments, they took on a negative charge under the conditions there extant. When a voltage was applied across the two compartments, the voltage dragged the charged DNA strand through the ion channel. The associated current showed the expected variations as the base pairs transited the ion channel.
Organic ion channels, however, have proven too delicate for use in a real gene sequencerxe2x80x94the haemolysin channels are quickly damaged by the stressful interactions involved. Additionally, the high compliance of the molecular structure of the ion channels reduces the distinctness of the signals characterizing the various base pairs. It was rapidly realized that a better choice would be a nanometer-scale aperture, or xe2x80x9cnanoporexe2x80x9d, made of some durable, hard, probably nonorganic material.
Unfortunately, there are no routine and controllable techniques available to fabricate such nanopores. Conventional technology can at best produce holes with diameters of 40-50 nm, whereas the requirement for a sequencing nanopore is roughly 2 nm.
A crude technique has been introduced to make small holes in silicon nitride. Schematically, this technique uses ion beam etching to make a shallow curved depression on each side of a substrate material, increasing the depth of the second depression until the two depressions meet, and thereby forming a small hole through the substrate. This technique has successfully been used to make holes as small as 4 nm, but the results of this type of process are extremely fragile, and also quite variable in nanopore size and shape.
There is a clear need for a technique to fabricate nanopores suitable for DNA sequencing, and for the product of such technique. The present invention enables fabrication of suitable nanopores in a silicon substrate by novel combination of well-known, controllable, and compatible microelectronic fabrication processes.
Techniques to fabricate nanoapertures with characteristic dimensions as small as 1-2 nm using novel silicon lithographic techniques has been developed. This is the only known technique to form apertures of this size. Such apertures are important for development of new approaches to DNA sequencing, and for other applications involving electronic, ionic, and molecular tunneling.